Lignocellulose is the most abundant component of biomass, comprising around half of plant matter produced by photosynthesis and representing the most plentiful renewable organic resource in the soil. It contains mainly three types of polymer: cellulose, hemicellulose and lignin. Each of these is strongly intermeshed and chemically bonded by non-covalent forces and by covalent crosslinkages. Cellulose accounts for up to 45% of total wood lignocellulose dry weight. It is a linear polymer composed of D-glucose subunits linked by β-1,4-glycosidic bonds forming long chains (or elemental fibrils) linked together by hydrogen bonds and van der Waals forces. Hemicelluloses are a heteropolymers representing, 15-35% of plant biomass and containing pentoses (β-D-xylose, α-L-arabinose), hexoses (β-D-mannose, β-D-glucose, α-D-galactose) and/or uronic acids. Lignin is composed of phenylpropane units joined together by different types of linkages. Lignin is linked to both hemicellulose and cellulose, coating them and forming a physical barrier between degrading enzymes and the plant cell wall.
In natural environments, cellulolytic microorganisms secrete enzymes that function synergistically, in association with the microorganism or independently. Although it is not fully known how many enzymes are involved in cell wall deconstruction, three general categories of enzymes are considered necessary to hydrolyze native cell wall materials: cellulases, hemicellulases and accessory enzymes such as hemicellulose debranching enzymes, phenolic acid esterase, and possibly lignin degrading and modifying enzymes. Three classes of enzymes act synergistically to hydrolyse cellulose: endo-β-1,4-glucanases, cleaving cellulose into shorter chains, cellobiohydrolases releasing cellobiose from the ends of the polymer, further cleaved into two glucose molecules by β-glucosidases. By loosening hemicellulose layers, hemicellulases presumably expose the cellulose fibers in the plant cell wall matrix, making them more accessible to hydrolysis. The efficient degradation of this polymer requires the concerted action of a range of enzymes working synergistically such as xylanases, β-mannanases, β-xylosidases, α-L-arabinofuranosidases and esterases. Lignin biodegradation by white-rot fungi is an oxidative process assisted by key enzymes such as phenoloxidase (laccase) and lignin-modifying peroxidase (lignin peroxidase, manganese peroxidase and versatile peroxidase).
The main industrial source of cellulases and hemicellulases is the mesophilic soft-rot fungus T. reesei (teleomorph Hypocrea jecorina), valued for the high protein secretion capacity of its mutant strains obtained by random mutagenesis (producing up to 100 g of extracellular protein per liter of culture).
In the context of biorefinery, chemical and structural features of biomass affect enzyme accessibility and activity, thereby affecting conversion costs. Pretreatment of lignocellulosic biomass aims to make the cellulose accessible to hydrolytic enzymes by altering the lignocellulosic cell wall. Pretreatment increases the accessible surface area, cellulose decrystallinization, partial cellulose depolymerization, hemicellulose and/or lignin solubilization, and the modification of the lignin structure.
Among fungal classes, basidiomycetes are known to be efficient degraders of cellulose, many species growing on dead wood or litter. The lignocellulolytic system of basidiomycetes has been studied intensively in the last decades. Genome sequencing and proteomic tools are often used, but the cellulolytic system is still not completely understood, especially the oxidative part of this system.
Cellobiose dehydrogenases (CDH; classified according to the enzyme nomenclature as E.C. 1.1.99.18; cellobiose: [acceptor] 1-oxidoreductase) are extracellular fungal hemoflavoenzymes produced by many white-rot fungi including Trametes versicolor, Phanerochaete chrysosporium, Ceriporiopsis subvermispora and P. cinnabarinus (Moukha, S. M. et al, 1999, Gene, vol. 234, pp: 23-33). CDH are also produced by the brown-rot fungus Coniophora puteana and the soft-rot fungus Humicola insolens. More recently, CDH from the ascomycetes Myriococcum thermophilum and Neurospora crassa were cloned and successfully expressed in Pichia pastoris. CDH are monomeric enzymes consisting of two prosthetic groups, a heme and a flavin domain. The heme-binding domain in the N-terminal position contains a cytochrome b-type heme which presents an unusual heme binding by Met/His ligation. The flavin domain in C-terminal binds FAD non-covalently and is classified as a member of the glucose-methano-choline family of oxidoreductases. These two regions are separated by a Thr-Ser-rich long linker region. The flavoprotein domain of CDH catalyzes two-electron oxidation of cellobiose and more generally cellodextrines, mannodextrines and lactose to corresponding lactones using electron acceptors such as dioxygen, quinones, phenoxyradicals and others. Also, one-electron transfer occurs. Heme is implicated in one internal electron transfer to FAD or another electron acceptor such as Fe3+.
Among oxidoreductases, laccases have been the most intensively studied, while CDHs are less well-researched. Only a few CDHs have been studied such as the CDH of P. chrysosporium, of Sclerotium (Athelia) rolfsii, or of Monilia sp.
Although the role of CDHs is still unclear, it is established that CDHs are produced in cellulolytic conditions and are involved in cellulose and lignin degradation. CDHs have been shown to bind cellulose by two different structures depending on species: a long aromatic-rich region for P. chrysosporium and P. cinnabarinus or a cellulose-binding domain for ascomycetes and soft-rot fungi, similar to that observed for cellulases (Henriksson G. et al, 1997, Journal of Biochemistry, vol. 324, pp: 833-838).
Their involvement in many reactions has been demonstrated, e.g. reduction of quinones, inhibition of phenol radical repolymerization, production of hydrogen peroxide, enhancement of manganese peroxidase turn-over and one of the most often cited reactions, the production of hydroxyl radicals by a Fenton-type reaction, which may participate in the degradation of cellulose, lignin and xylan. CDHs are known to enhance the action of cellulases on crystalline cellulose and also to degrade wood components, but their role in complex lignocellulosic substrate degradation has never been investigated.
In 2008, various compositions comprising T. reesei cellulases combined with CDHs from different origins were tested in the International application WO2010/080532 which demonstrated that, unless to add a Family 61 glycoside hydrolase, each of the tested compositions comprising T. reesei cellulases combined with CDHs from different origins led to an inhibition of the crystalline cellulose degradation, therefore not enabling an improved degradation of lignocellulosic biomass.